Application of pharmaceutical preparation containing BAY 73-6691 or derivative thereof in preparation of drugs for treating brain injury diseases

Abstract

The invention belongs to the field of biological medicines, and relates to application of a pharmaceutical preparation containing BAY 73-6691 or a derivative thereof in preparation of a medicine for treating brain injury diseases. Wherein the structural formula of the BAY 73-6691 is as follows: # imgabs0 #. The pharmaceutical preparation comprises the BAY 73 to 6691 and / or a pharmaceutically acceptable salt of the BAY 73 to 6691. According to the present invention, the research results show that the BAY 73-6691 can improve the ARCA brain injury through the ubiquitin phosphorylation dual pathway, such that the BAY 73-6691 may become the ARCA treatment candidate drug; the application further explains a new drug mechanism and an action target spot of the BAY 73-6691; meanwhile, a new supplement is expected to be provided in clinical application of new use of old medicines.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of a pharmaceutical preparation containing BAY 73-6691 or its derivatives in the preparation of drugs for treating brain injury diseases. Background Art

[0002] Autosomal recessive cerebellar ataxia (ARCA) is a group of clinically and genetically highly heterogeneous central nervous system diseases characterized by progressive ataxia, which are characterized by adult-onset. They mainly cause cerebellar degeneration and atrophy, leading to balance, coordination and gait disorders, or are accompanied by other clinical symptoms such as cognitive impairment and dementia. The current prevalence rate in the population is about 4 in 100,000. Most patients have a family history, and a few patients are sporadic. The characteristic pathological changes are the degeneration and death of cerebellar Purkinje cells. Gordon Holmes Syndrome (GHS [OMIM 212840]) is a subtype of ARCA, and the main clinical manifestations are ataxia symptoms that begin in adolescence, combined with gonadal dysfunction. Earlier, we reported for the first time in humans that mutations in the CHIP protein-encoding gene STUB1 can cause GHS, proving that STUB1 is the third pathogenic gene of GHS. Subsequent molecular genetics and animal model studies suggest that biallelic CHIP mutations leading to CHIP protein dysfunction are an important cause of ARCA. Similar to the human phenotype, the CHIP mutation model also exhibits progressive clinical manifestations, mainly cerebellar degeneration and atrophy, leading to ataxia symptoms such as balance, coordination and gait disorders, and accompanied by other cognitive disorders and dementia phenotypes. At present, the clinical treatment of ARCA is extremely limited, and its mortality and disability rates are extremely high. The first choice in clinical practice can only be supportive and symptomatic treatment. Therefore, there is an urgent need to further explore the pathogenic mechanism of this type of disease and find new treatment and neuroprotective targets.

[0003] STIP1 homology and U-box containing protein 1 (STUB1), also known as Carboxyterminus of Hsp70-interacting protein (CHIP), is encoded by the CHIP / STUB1 gene and is located on chromosome 16 of the human genome. The functional domains of CHIP protein mainly include the tetratricopeptide repeat (TPR) domain at the amino terminus that mediates the molecular chaperone function, the coiled coil (CG) domain that promotes the formation of protein dimers, and the U-box domain at the carboxyl terminus that has E3 ubiquitin ligase activity. The TPR domain at the N terminus of the CHIP protein plays a molecular chaperone function, which mainly binds to heat shock proteins HSP70 or HSP90. HSP-CHIP can connect to substrate proteins, complete targeted modification of substrate proteins, inhibit the folding activity of chaperone proteins, and promote the physiological decomposition and recycling of proteins. The U-box domain confers CHIP ubiquitin ligase activity, which is responsible for recruiting ubiquitin-binding enzyme E2. Through the cascade reaction of E1 ubiquitin activating enzyme-E2 ubiquitin-binding enzyme-CHIP, the ubiquitin bound to E2 is transferred to the chaperone molecule bound to the TPR domain and its substrate protein or directly to the substrate protein bound to CHIP, thereby forming a polyubiquitin chain on the substrate. Due to the dual properties of molecular chaperone and E3 ligase, CHIP plays an important role in the protein quality control system (Protein quality control, PQC). CHIP can maintain normal cell function by promoting molecular chaperone-mediated degradation of abnormal proteins through proteasomes and autophagy-lysosome-dependent pathways. Animal models using CHIP genetic loss of function models have reported worsening of the heart's response to hemodynamic or ischemic stress, accelerated aging and neurodegenerative diseases.

[0004] CHIP protein mutations have been confirmed to be the cause of ARCA, so it belongs to CHIP-related ataxia. Through brain proteomic studies of CHIP-related hereditary ataxia animal models, it was found that the expression of PDE9A in the brain tissue of CHIP-related hereditary ataxia animal disease models was significantly increased, and its expression level increased progressively with the progression of the disease, suggesting that PDE9A may be involved in the pathogenesis of CHIP-related ataxia. Phosphodiesterases (PDEs) are a super enzyme family responsible for hydrolyzing the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). According to their structural and functional characteristics, PDE can be divided into 11 subfamilies (PDE1-PDE11). Among all PDE subfamilies, phosphodiesterase 9A (PDE9A) has the highest affinity for cGMP and is highly expressed in the cortex, hippocampus, basal ganglia and cerebellum. Moreover, PDE9A is the most highly expressed PDE subtype in brain tissue and is widely involved in cell signaling pathways through cGMP. cGMP signaling is important for many functions in the brain, such as synaptic plasticity, phototransduction, learning and memory, and stem cell differentiation. Therefore, upregulation of PDE9A expression may inhibit cell signaling pathways regulated by cGMP. Inhibition of PDE9A activity has been reported in a variety of diseases including AD. Inhibition of PDE9A can not only reduce the oxidative damage of hippocampal neurons caused by Aβ deposition, but also has potential therapeutic effects on animal models of various diseases such as myocardial damage caused by heart failure and DMD.

[0005] As an emerging class of drugs, PDE9A inhibitors are in the active research and development stage. There may be no drugs approved for clinical treatment on the market at present. However, in the research and development stage, these compounds are being explored in preclinical or clinical trials for the treatment of various diseases, especially neurodegenerative diseases. Among them, PF-04447943 has entered Phase II clinical trials for Alzheimer's disease; BI 409306 has entered Phase II clinical trials for Alzheimer's disease and Phase II clinical trials for schizophrenia; and the earliest discovered BAY 73-6691 is still in the early stages of preclinical research. As a widely used PDE9A inhibitor, BAY 73-6691 prevents the decomposition of cGMP by inhibiting PDE9A, thereby increasing the level of cGMP. BAY 73-6691 is designed to highly selectively target PDE9A, so it has less inhibitory effect on other PDE family members. This high selectivity helps to reduce nonspecific drug effects and side effects. Previously, in animal models, BAY 73-6691 was reported to have potential effects on cognitive function and therefore may have an ameliorative effect on Alzheimer's disease.

[0006] However, the drug development process of BAY 73-6691 is complex and faces great challenges in its translation into clinical applications. To date, detailed data on pharmacokinetics, bioavailability and in vivo stability are very limited, and its pharmacological effects are still relatively blank. Therefore, it is not clear what diseases BAY 73-6691 will have a therapeutic effect on as a drug. Summary of the invention

[0007] In order to further study the therapeutic effect of BAY 73-6691 and the correlation between BAY 73-6691 and ARCA brain injury, the present invention provides a use of a pharmaceutical preparation containing BAY 73-6691 or a derivative thereof in the preparation of a drug for treating brain injury diseases.

[0008] The technical solution of the present invention is achieved in this way:

[0009] Use of a pharmaceutical preparation containing BAY 73-6691 or its derivatives in the preparation of a drug for treating brain injury diseases, wherein the structural formula of BAY 73-6691 is:

[0010] The above-mentioned pharmaceutical preparations include BAY 73-6691 and / or a pharmaceutically acceptable salt of BAY 73-6691.

[0011] The salt in the pharmaceutically acceptable salt of BAY 73-6691 is any one of hydrochloride, hydrobromide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, propionate, hexanoate, heptanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, maleate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, p-toluenesulfonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, dodecyl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate and stearate.

[0012] The above-mentioned brain injury diseases are secondary brain injuries caused by ARCA.

[0013] The above-mentioned brain damage disease is autosomal recessive spinocerebellar ataxia.

[0014] The above-mentioned CHIP inactivation mutation will lead to increased PDE9A expression in ARCA brain tissue, and the PDE9A content will progressively increase with the progression of the disease.

[0015] The above-mentioned autosomal recessive spinocerebellar ataxia is caused by impaired mitochondrial autophagy in Purkinje cells.

[0016] The present invention has the following beneficial effects:

[0017] 1. This study found that BAY 73-6691 can improve ARCA brain damage through the dual pathway of ubiquitin phosphorylation. Therefore, BAY 73-6691 may become a candidate drug for the treatment of ARCA; at the same time, it is expected to provide new supplements for the clinical application of old drugs in new uses.

[0018] 2. BAY 73-6691 regulates the cGMP / cAMP pathway in the central nervous system and participates in regulating the CHIP-related protein quality control system, explaining the new pharmacological mechanism and target of BAY 73-6691.

[0019] 3. BAY 73-6691 inhibits PDE9A activity to maintain the balance of cGMP / cAMP signal interaction mediated by CHIP, and improves the pathological changes of ARCA brain damage caused by mitochondrial autophagy disorders in Purkinje cells. Improving the imbalance of intracellular cGMP / cAMP pathways has potential therapeutic and brain-protective effects on ARCA. It can also enhance the clearance of damaged / misfolded proteins and inhibit the ability of protein water toxicity, as well as immunity, aging, metabolic stress, and many human diseases, including heart and nervous system diseases.

[0020] 4. BAY 73-6691 inhibits the increase of PDE9A aggregation and increases the expression of CHIP protein to improve mitochondrial damage and maintain the balance of mitochondrial autophagy; it treats the clinical phenotype of ARCA by inhibiting the toxic damage mediated by PDE9A protein aggregation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1The two-way degradation regulation of PDE9A and CHIP protein; a: 4ug of Flag-CHIP plasmid was co-transfected with 0ug, 4ug, 8ug, and 16ug of V5-His-PDE9A into 293T cells, and western blot was used to analyze the expression of PDE9A regulated by CHIP. b: 4ug of V5-His-PDE9A plasmid was co-transfected with 0ug, 4ug, 8ug, and 16ug of Flag-CHIP into 293T cells, and western blot was used to analyze the changes of PDE9A mediated by CHIP. c: Changes in PDE9A mRNA after cells were transfected with different mutant plasmids of CHI P, T246M: homozygous mutation at p.T246M, K30A: homozygous mutation at p.K30A, CHIP+Bay: cells overexpressed CHIP and then added 200ug / ml BAY 73-6691 (PDE9A inhibitor), sh-CHIP-1, sh-CHIP-2 and sh-CHIP-3: all cells interfered with CHIP expression; d: Changes in PDE9A after cells were transfected with CHIP mutant plasmids: e: Changes in CHIP mRNA after cells were transfected with PDE 9A mutant plasmids; PDE9A+Bay cells overexpressed PDE9A and then added BAY 73-6691, sh-CHIP: cells interfered with CHIP expression; f: Changes in PDE9A after changes in PDE9A protein expression; g: cells were transfected with HA-CHIP and Flag-PDE9A plasmids, 50nM BafilomycinA1 (autophagy inhibitor) and 100nM Bozteomib (protease inhibitor, PS-341) were used to detect the half-life and degradation pathway of CHIP and PDE9A proteins; h: After HA-CHIP and Flag-PDE9A were co-transfected into cells, the bidirectional degradation regulation of CHIP and PDE9A was observed; i: The degradation effect of PDE9A, CHIP and BAY 73-6691 on PDE9A and CHIP proteins; g: Schematic diagram of the structure of PDE9A and CHIP proteins, ΔTPR: TRP domain knockout, ΔU-box: U-box domain knockout; k: Immunoprecipitation reaction to detect the interaction domain between PDE9A and CHIP proteins. Antibody: V5-rabbit, Flag-mouse; I: PDE9A and CHIP plasmids were co-transfected into 293T cells, and immunofluorescence was used to observe the co-localization of PDE9A and CHIP in the cytoplasm. Data are expressed as mean ± standard deviation, ANOVA analysis (Tukey's multiple comparison), ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, ns P>0.05.

[0023] Figure 2The predicted model of PDE9A and HSP70 molecular docking; a: β-SBD domain of HSP70 binds to PDE9A, NBD: N-terminal nucleotide binding domain; β-SBD: β-sandwich substrate binding subdomain; α-SBD: C-terminal α-helical lid. b: Top1 predicted model of β-SBD hydrogen-bonded to PDE9A. The CYS338 site of PDE9A and the TYR183 site of HSP70 are connected by hydrogen bonds. The possible model was predicted by gram-x software (http: / / vakser.bioinformatics.ku.edu / resources / gramm / grammx). The prediction results of PDBePISA and dockeasy were visualized (www.dockeasy.cn).

[0024] Figure 3 CHIP directly mediates K63 and K27-linked PDE9A polyubiquitination; a: schematic diagram of ubiquitin mutant structure. b: schematic diagram of homozygous mutation of CHIP gene p.T246M site and p.K30A site; c: in vitro ubiquitination experiment, E1, E2: UbcH5, ATP: 1XATP buffer; d: CHIP-mediated ubiquitination modification of PDE9A protein by different E2 enzymes. Ube2V-Ubc1, UbcH5D and UbcH5a can all synthesize ubiquitinated chains in vitro; e: CHIP and PDE9A in vivo ubiquitination experiment, Antibody: V5-rabbit, HA-mouse; f and g: In vivo ubiquitination experiments verify that different ubiquitin chain mutants participate in CHIP ubiquitination modification of PDE9A; K4, K6, K1, K2, K29 chains all produce polyubiquitin chains, while K63R and K27R ubiquitin chain synthesis is impaired; f: In vivo ubiquitination experiment detects the structure of CHIP ubiquitination modification of PDE9A, and CHIP domain deletion has a barrier to ubiquitination chain synthesis; g: In vivo ubiquitination experiments show that CHIP p.T246M and p.K30A mutations prevented the ubiquitination of PDE9A; j: cAMP activator Forskolin (50uM) was added to the K63 and K27-linked CHIP-mediated PDE9A ubiquitination in vivo, and the in vitro ubiquitination assay showed that the ubiquitination reaction of CHIP to PDE9A changed after cAMP activation.

[0025] Figure 4To inhibit PDE9A activity and increase exogenous CHIP protein to improve the clinical phenotype of ARCA; a: Timeline of behavioral tests in the BAY 73-6691 treatment group and the AAV-CHIP injection group. The BAY 73-6691 treatment group included: BAY 73-6691, wild-type littermates, homozygous Hom mutant and homozygous mutant 4-month-old male rats; the AAV-CHIP injection group included: AAV-CHIP, wild-type littermates, Hom and AAV-NC male rats injected into the same littermates; b and j: The maximum speed tolerated by the running wheel in the rat running wheel test. c and k: The time required for rats to completely pass the balance beam in the balance beam test; d and l: Gait analysis test shows rat footprints, red for forelimbs and blue for hindlimbs. LFSL: left front step length, RFSL: right front step length, LRSL: left rear step length, RRSL: right rear step length, LFRO: left front-to-back overlap, RFRO: right front-to-back overlap, FBW: front-base widt h, RBW: rear-base width, e and m: quantitative analysis of rat gait; f and n: trajectory diagram of water maze experiment, trajectory diagram of rats exploring the platform after entering the water from the third quadrant after hiding the platform; g and o: after hiding the platform, the latency of rats entering the water from the third quadrant to explore the platform was monitored for 5 consecutive days; h and p: on the sixth day of water maze, after removing the platform, the proportion of time the rats stayed in the quadrant after entering the water from the third quadrant; i: after removing the platform, the number of times the rats crossed the platform within 60 seconds after entering the water from the third quadrant; q: after removing the platform, the number of times the rats crossed the platform within 5 minutes after entering the water from the third quadrant; data are expressed as mean ± standard deviation, ANOVA analysis (Tukey's multiple comparisons), ****P<0.001, ***P<0.01, **P<0.02, *P<0.05, ns P>0.05.

[0026] Figure 5 PDE9A inhibits CHIP Serine 20 phosphorylation; a: Phospho S20 antibody was used to detect CHIP phosphorylation in the cerebellum and hippocampus of male rats in the Bay group and the wild-type and Hom groups; b: CHIP phosphorylation levels in the cerebellum and hippocampus of male rats in the AAV-CHIP, AAV-NC, wild-type and Hom groups; c: CHIP mutant plasmids and different PDE9A expression plasmids were transfected into 293T cells to change the CHIP expression level, and western blot was used to detect the CHIP phosphorylation level. Data are expressed as mean ± standard deviation, ANOVA analysis (Tukey's multiple comparisons), ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, ns P>0.05.

[0027] Figure 6To detect the toxic damage of PDE9A aggregation and CHIP to cells; a: AnnexinV / PI flow apoptosis experiment was used to detect the apoptosis of 293T cells when the expression of PDE9A and CHIP proteins changed. AnnexinV was used to detect early cell apoptosis, and the nucleic acid dye PI was used to detect late apoptosis because it penetrates the cell membrane. Q1 was mechanical necrotic cells, Q2 was late apoptotic cells, Q3 was normal cells, Q4 was early apoptotic cells, and the sum of Q2+Q4 was selected for quantitative statistics of apoptosis; b: CCK8 experiment was used to detect the cell proliferation activity when the expression of PDE9A and CHIP proteins changed. After adding CCK8 reagent to each group, the absorbance was measured after incubation at 37℃ for 2h, and the detection time gradient was selected as 24h, 48h, 72h, 96h, and 120h.

[0028] Figure 7 BAY 73-6691 and exogenous CHIP improve ARCA brain tissue pathology; a and b: tissue immunofluorescence quantification of cerebellar tissue Purkinje cell marker protein calbindin-D28K, to determine the changes in the number of cerebellar Purkinje cells; c and d: fully automated capillary electrophoresis to detect the expression of autophagy proteins LC3B and P62 in the cerebellum and hippocampus of each group of mice; e: transmission electron microscopy observation of mitochondrial changes in rat cerebellum tissue. Ho m group mitochondria (M, red dotted circle) were less in number, membrane was partially blurred and damaged, cristae structure was broken and dissolved in large quantities, matrix was dissolved in large quantities or vacuolated, and autophagolysosomes (ASS, red arrows) were visible under the microscope; N: nucleus, Go: Golgi apparatus, Lip: lipofuscin, RER: rough endoplasmic reticulum, AP: autophagosome. Data are expressed as mean ± standard deviation, ANOVA analysis (Tukey's multiple comparison), ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, ns P>0.05.

[0029] Figure 8 The effects of PDE9A aggregation and CHIP reduction on the structure of hippocampal neurons were investigated in rats with STUB1 p.T246M homozygous mutation (Hom, age 6.25 months), rats exogenously administered CHIP by adeno-associated virus (AAV-CHIP, age 7 months), rats inhibited PDE9A activity by BAY 73-6691 (Bay, age 6.25 months), and male rats of the same age and littermate (Wt, age 6.25 months). The hippocampal neurons of each group were observed by Nissl staining. In the Wt, Bay, and AAV-CHIP groups, more Nissl bodies were observed, which were arranged in an orderly manner and the cell nuclei were clearly located in the center; in contrast, the number of neurons in the CA1 region of the hippocampus in the Hom group was reduced, the arrangement was disordered, the number of Nissl bodies was reduced, and the cell nuclei were missing or displaced.

[0030] Fig. 9 Single-cell sequencing cell clustering; a: Schematic diagram of single-cell transcriptome analysis. There are 4 model groups, three in each group: STUB1 p.T246M homozygous mutant rats (Hom, 6.25 months old), exogenously injected AAV-CHIP rats (AAV-CHIP, 7 months old), PDE9A inhibitor BAY 73-6691 treated rats (Bay, 6.25 months old), and littermate wild-type male rats (Wt, 6.25 months old); b and c: Cell clustering diagrams, according to the marker genes of rat hippocampal and cerebellar cells, the cells are divided into 12 groups: granule cells (Reln, Rbfox3, Neurod1), oligodendrocytes (Mpb), glutamatergic neurons (Aldoc), GABAergic neurons (Gad2, Pnoc, Tcf7l2, Lhx9), GABA interergic neurons (Pax2, Lbx1), pyramidal cells (N ell1, Npy2r, Slc4a8, B3gat2), oligodendrocyte precursor cells (Pdgfra, Olig1, Olig2), endothelial cells (C1qa, C1qb, C1qc), Bergmann glial cells (Gdf10, S100b), glial cells (Tmem119), Purkinje cells (Gad1, Car8, Calb1, Pcp2, Necab2, Pcp4), astrocytes (Clu, Apoe); d: volcano plot showing that 12 groups of cells express specific marker genes.

[0031] Fig.10 PDE9A participates in CHIP-mediated cGMP / cAMP signaling interaction; ac: KEGG enrichment analysis of differentially expressed genes in total cells of Wt vs Hom, Bay vs Hom and AAV-CHIP vs Hom was performed, and the red box selected the most relevant signaling pathway of ARCA. d: Aucell pathway activity analysis showed the expression of cGMP-PKG pathway in total cells of 4 groups of rats. eg: GSEA pathway activity analysis showed the changes of cGMP-PKG signaling pathway in total cells of Wt vs Hom, Bay vs Hom and AAV-CHIP vs Hom; h: Aucell pathway activity analysis showed the expression of cAMP signaling pathway in 4 groups of rats. ik: GSEA analyzed the changes of cAMP signaling pathway in total cells of Wt vs Hom, Bay vs Hom and AAV-CHIP vs Hom.

[0032] Fig.11Bidirectional post-translational modification of PDE9A and CHIP; ac: KEGG enrichment analysis of differentially expressed genes in Purkinje cells between Wt vs Hom, Bay vs Hom and AAV-CHIP vs Hom, with the most relevant signaling pathways selected in red. di: Aucell pathway activity analysis showed changes in cGMP-PKG signaling pathway, ubiquitin-mediated proteolysis, mitochondrial autophagy, cAMP signaling pathway, oxidative phosphorylation and autophagy signaling pathways in Purkinje cells of the four groups of rats.

[0033] Fig.12 The expression of pathway signaling proteins; a and b: Western blot detection of cAMP-PKA signaling protein expression in the cerebellum and hippocampus of each group of mice; d: ELISA detection of cGMP content in the cerebellum and hippocampus of male rats in the wild-type and Hom groups injected with BAY 73-6691 intraperitoneally and the same littermate; c: cAMP activator Forskolin (50uM) was added to the K63- and K27-linked CHIP-mediated PDE9A ubiquitination in vivo, and the in vitro ubiquitination assay showed that after activating cAMP, the ubiquitination reaction of CHIP to PDE9A changed. e: ELISA was used to detect the cGMP content in the cerebellum and hippocampus of male rats injected with AAV-CHIP in the tail vein, wild-type mice, Hom group and homozygous mutant mice in the same littermates and injected with empty AAV (AAV-NC) in the tail vein; f and g: Automatic capillary electrophoresis was used to detect the expression of PKG signaling protein in the cerebellum and hippocampus of mice in each group; data are expressed as mean ± standard deviation, ANOVA analysis (Tukey's multiple comparison), ****P < 0.001, ***P < 0.01, **P < 0.02, *P < 0.05, ns P>0.05.

[0034] Fig.13 This is a schematic diagram of the main results of this study. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Experimental Materials:

[0037] Plasmids and antibodies

[0038] All plasmids in this experiment were purchased from Shanghai GeneCare Gene Co., Ltd.:

[0039] pCMV-PDE9A-3xflag; pCMV-PDE9A-eGFP; pCMV-PDE9A-V5-6his; pCMV-PDE9A(del236-557aa)-V5-6his; pCMV-STUB1-3xflag; pCMV-STUB1-HA; pCMV-STUB1(del226-300aa)-3xflag; pCMV-STUB 1(del26-aa127)-3xflag; pCMV-STUB1(T246M)-3xflag; pCMV-STUB1(K30A)-3xflag; pCMV-HA-ubiquitin-WT; pCMV-HA-ubiquitin-K48; pCMV-HA-ubiquitin-K48R;

[0040] pCMV-HA-ubiquitin-K63;

[0041] pCMV-HA-ubiquitin-K63R;

[0042] pCMV-HA-ubiquitin-K11;

[0043] pCMV-HA-ubiquitin-K11R; pCMV-HA-ubiquitin-K27; pCMV-HA-ubiquitin-K27R; pCMV-HA-ubiquitin-K29; pCMV-HA-ubiquitin-K29R; pCMV-HA-ubiquitin-K0; shRNA-PDE9A; shRNA-STUB1-1; shRNA-STUB 1-2; shRNA-STUB1-3。

[0044] List of antibodies:

[0045] Anti-PDE9A-rabbit (proteintech, China); Anti-PDE9A-mouse (santacruz, USA); anti-STUB1-rabbit (abcam, USA); anti-STUB1-mouse (santacruz, USA); anti-HA-tag-mouse (abcam, USA); anti-DYKDDD DK-mouse (proteintech, China); anti-V5-rabbit (proteintech, China); anti-GFP-tag-rabbit (abcam, USA); anti-ubiquitin-mouse (abcam, USA); anti-PRKG1-rabbit (proteintech, USA); anti-PRKG2-rabbit (proteintech, China); anti-CREB1-mouse (proteintech, China); anti-cAMP-Rabbit (proteintech, China); anti-PRKA CA-Rabbit (Proteintech, China); Anti-Calbindin-rabbit (Abcam, USA); anti-GAPDH-mouse (Proteintech, China); anti-pho-CHIP (Abmart, China); HRP-conjugatedAffinipure Goat Anti-Rabbit IgG (H+L) (Protei ntech, China); HRP-conjugatedAffinipure Goat Anti-mouse IgG (H+L) (Protei ntech, China); Goat Anti-Rabbit IgGH&L ( 488)(abcam, USA); Goat Anti-mouse IgGH&L( 594)(abc am, united states).

[0046] Cell culture and plasmid transfection

[0047] HEK293T cells (cell bank of Chinese Academy of Sciences) were cultured in DMEM high glucose medium mixed with 10% fetal bovine serum (Hyclone, China). All plasmids were transfected into 293T cells according to the instructions of lp3000 transfection kit (Thermo Fisher Scientific, USA). The medium was changed 6 hours after transfection. The medium change included adding 20uM of BafilomycinA1, Bozteomib protease inhibitor, and 200ug / ul of PD E9A inhibitor BAY 73-6691 to the medium. The medium was removed at specific times for further analysis.

[0048] Experimental Animal Care and Treatment

[0049] All animal experimental operations and treatments in this study were approved by the Ethics Committee of the Animal Experiment Center of Zhengzhou University. The animals were bred and raised in the Animal Center of Zhengzhou University under specific pathogen-free (SPF). The animals used in this experiment were the offspring of CHIP p.T246M mutant rats from previous experiments. Rat tail DNA was extracted using a kit and

[0050] CHIP positive strand: 5'-CTCATGGGCAGGCTCTGGTATGG-3',

[0051] PCR reaction was carried out with primer 5'-GAGCAGTTCAGAACCCATCATCAGG-3' of the antisense strand of CHIP, and the product was sequenced by Sanger in Shangya Company for genotype identification. +15 Month-old male mutant rats were intraperitoneally injected with 200ug / kg of protease inhibitor BAY 73-6691 daily for 15 consecutive days, and subsequent experiments were performed 7 days later. Another 5-month-old male homozygous mutant rats were selected for exogenous overexpression of CHIP in brain tissue. The rats were anesthetized by inhalation of 1.8% isoflurane and injected with 1.2*10 12 vg / ml HBAAV2 / BBB-C HIP virus (Hanbi Biotechnology, China) 100ul was added and the rats were continued to be raised. Male homozygous mutant rats from the same litter were injected with the same dose of HBAAV2 / BBB-flag as a control, and subsequent experiments were performed when the rats were 7 months old; the control group included male normal wild rats and homozygous mutant rats from the same litter.

[0052] Monocyte RNA sequencing and biological information analysis

[0053] Rats in the PDE9A inhibitor treatment group (6 +7 Months old), male normal wild rats of the same litter (6 +7 Months old), homozygous mutant rats (6+7 Three rats were included in each of the HBAAV2 / BBB-CHIP group (7 months old) and the intravenously injected HBAAV2 / BBB-CHIP group (7 months old). After 20 minutes of inhalation anesthesia with 1.8% isoflurane, fresh rat hippocampus and cerebellum tissues were quickly extracted. After single-cell sorting at Nanjing Xingeyuan Company, three samples in each group were mixed with hippocampal neurons and cerebellar neurons in a 1:1 ratio to capture mRNA to construct a single-cell library. The library sequencing was completed by Wuhan BGI Co., Ltd. using the MGISEQ-2000 platform PE150 sequencing strategy; the sequencing results were compared with the original sequencing files in Fastq format, and the 10X format single-cell gene expression matrix was obtained by processing the original sequencing files in Fastq format using the CeleScope standardized process. The scRNA-seq data set was further processed and analyzed in R software. The data was first quality controlled, and cells with unique gene expression exceeding 2500 or less than 500, mitochondrial counts>15% and gene coverage cells<3 were filtered, that is, empty droplet cells and low-quality cells contaminated by mitochondria were eliminated; then the data was normalized, using the SCTransform function for standardization, and harmony (v.0.1.0) software to remove batch effects between samples; Seurat was further used for dimensionality reduction, clustering, grouping, and visualization of marker genes to annotate cell subpopulations. Finally, FindMarkers was used to calculate differentially expressed genes, with a threshold of log2FC>0.25, p<0.05, and a two-sample T test. The differentially expressed genes were further analyzed by KEGG enrichment, and the KEGG Pathway and Moduledata of rattus norvegicus (rat) came from ClusterProfiler software; GSEA analysis identified the expression trend of reference gene sets between groups; AUCell was used for single-cell pathway activity analysis.

[0054] Real-time fluorescent quantitative polymerase chain reaction

[0055] RNA was extracted using the RNA extraction kit and Prime Script TM RT reagent Kit with gDNA Eraser (Takara, Japan) reverse transcription kit was used to reverse transcribe RNA into cDNA according to the instructions. After the cDNA was diluted 20 times, the mRNA expression of CHIP and PDE9A in different groups of cells was detected by combining CHIP and PDE9A primers. Real-time fluorescence quantitative polymerase chain reaction was performed using the RT-qPCR kit according to the instructions, with three replicates per group. Primers included:

[0056] CHIP sense strand: 5′-ATCCCCGACTACCTGTGTGGC-3′ and antisense strand: 5′-TGCTCCTCGATGTCCTTGCG-3′;

[0057] PDE9A sense strand: 5′-TTGACTCCTCGACGCGATGTTC-3′ and antisense strand: 5′-CTGAGGGTGACAGGGTTGATGC-3′;

[0058] GAPDH sense strand: 5′-GGCGCTGAGTACGTCGTGGAGT-3′ and antisense strand: 5′-GGGCAGAGATGATGACCCTTTTG-3′.

[0059] Western blotting

[0060] Take a certain amount of rat brain tissue, hippocampal tissue and collected cells, add RIPA lysis buffer, PMSF, protease inhibitors, phosphatase inhibitors in a ratio of 1000:10:10:10, then fully lyse the cells in an ultrasonic lyser to prepare protein samples, and unify the protein concentration of each group by BCA protein quantification method. For western blotting, dilute the protein samples with 5XSDS protein loading buffer and then heat at 100℃ for 5min. Use BeyoGel TM Plus4-20%PrecastPAGE Gel for Tris-Gly System with 15well (Beyotime, China) was used for protein electrophoresis and membrane transfer. The primary antibodies were selected as protein markers and incubated overnight at 4℃ (Flag-mouse 1:5000, PDE9A-mouse 1:5000, CHIP-rabbit 1:10000, v51:5000, HA 1:20000, ubiquitin 1:1000, cAMP 1:2000, PRCAKA 1:1000, GAPDH 1:5000, β-tubulin 1:5000). The next day, the membrane was washed and incubated with secondary antibodies (HRP-conjugated Affinipure Goat Anti-Rabbit 1:5000, HRP-conjugated Affinipure Goat Anti-mouse1: 5000), add ECL chemiluminescent A+b mixed solution and develop under an exposure instrument;

[0061] The final concentration of protein samples in each well of the fully automated capillary electrophoresis experiment was 2 ug / ul. Standard operations were performed according to the instructions of the Chemiluminescence training Kit (ProteinSimple, USA) and completed in SimpleWestern Jess (ProteinSimple). The protein cartridge was 25, the size range was 12-230 kDa, and the primary antibody was diluted to a specific concentration (PDE9A-rabbit 1:50, CHIP-rabbit 1:100, PRKG1-rabbit 1:30, PRKG2-rabbit 1:25, P62-rabbit 1:25, LC3B-rabbit 1:40, β-tubulin-rabbit 1:100).

[0062] ELISA

[0063] The same concentration of mouse and hippocampal tissue protein lysate was used in each group, and the cGMP content in the tissue was quantitatively detected using Cyclic GMP Complete ELISA Kit (abcam, USA).

[0064] Protein half-life determination

[0065] 293T cells transfected with CHIP-HA alone, PDE9A-flag alone, or co-transfected with CHIP and PDE9A plasmids were replaced with culture medium containing different reagents for 24 hours, and 40uM cycloheximide was added. Cells were collected at different time points of 0h, 2h, 4h, 6h, 8h, and 10h for western blotting to detect the expression of PDE9A and CHIP proteins.

[0066] Co-immunoprecipitation

[0067] The 293T cells transfected with plasmids were collected and lysed with NP-40 lysis buffer (Beyotime, China), PMSF, protease inhibitors and phosphatase inhibitor mixture in a ratio of 1000:10:10:10 to obtain a 5ug protein sample. TMProtein A+G beads (Beyotime, China) were added for 1 hour, and the magnetic beads were discarded. The labeled protein antibodies (V5-rabbit 10ug, GFP-rabbit 10ug) were added and incubated at 4°C for 12 hours on a side-swing shaker. Then, new 100ul magnetic bead mixture was added and incubated at 4°C overnight. After 12 hours, the magnetic beads were washed 4 times with NP-40 lysis mixture for 5 minutes, and then 20ul of 5XSDS protein loading buffer and 30ul of NP-40 lysis mixture were added. After heating at 100°C for 5 minutes, the supernatant was taken and the target protein was detected by western blotting.

[0068] Immunostaining

[0069] Cell immunofluorescence: cells were plated in 24-well plates, fixed with 4% paraformaldehyde (Beyotime, China) for 15 min, permeabilized with 0.3% TritonX-100 (PBS) (Beyotime, China) for 20 min, blocked with 5% fetal bovine serum albumin (PBS) (Beyotime, China) for 1.5 h, incubated with primary antibodies at 4°C overnight (CHIP-mouse 1:500, PDE9A-rabbit 1:500), and incubated with fluorescent secondary antibodies at room temperature for 1 h ( 488 1:500, 594 1:500), and DAPI (Beyotime, China) was added and the slides were sealed.

[0070] Tissue immunofluorescence: Rats were anesthetized by inhalation of 1.8% isoflurane for 20 minutes. The brain was removed by cardiac perfusion with 4% paraformaldehyde, and the sections were embedded in paraffin with a thickness of 4um. First, the brain was dewaxed with environmentally friendly dewaxing solution for 30 minutes, and then dehydrated with gradient alcohol (100% I, 100% II, 95%, 85%, 75% for 5 minutes each), and then antigen repair was performed with 50X antigen repair solution. The cells were permeabilized with 0.3% TritonX-100 (PBS) at 37°C for 20 minutes, blocked with 5% BSA (PBS) for 1.5 hours, and incubated with primary antibodies at 4°C (Calbindin-rabbit 1:300, CHIP-mouse 1:500, PDE9A-rabbit 1:500). After 12 hours, the fluorescent secondary antibody was incubated at room temperature for 1 hour ( 488 1:500, 594 1:500), DAPI was added to cover the slides, and fluorescence images were taken under an automatic fluorescence microscope (Leica, Germany).

[0071] In vivo and in vitro ubiquitination assays

[0072] In vivo ubiquitination: CHIP-flag, PDE9A-v5-his, HA-ubiquitin (the above human ubiquitination wild type and mutant plasmids) plasmids were transfected into 293T cells, and COIP was performed by combining V5 antibody (v5-rabbit10ug) with magnetic beads. The primary antibodies for western blotting analysis included (flag-mouse 1:5000, HA-mouse 1:2500, ubiquitin-mouse 1:1000).

[0073] In vitro ubiquitination: In vitro ubiquitination experiments were performed using 0.3uM E1, 3.4uM E2, 250uM Ubiquitin, 10XATP assay buffer (200mM HEPES-KOH, 50mM MgCl2, 20mM ATP, 5mM DTT) (RD, USA) to prepare a 10ul system, incubated at 37°C for 1h, boiled after adding SDS, and detected by WB. E2 ligases were selected from Ube2V-Ubc13, UbcH5D, and UbcH5a (RD, USA).

[0074] Cell proliferation and cytotoxicity (CCK8 assay)

[0075] 1000 cells were seeded in each well of a 96-well plate, and the cell proliferation activity was detected at 24 h, 48 h, 72 h, 96 h, and 120 h using Cell Counting Kit-8 (Dojindo, Japan) according to the instructions.

[0076] Flow cytometry

[0077] Get 1x10 per group 6 1x10 cells were filtered through a cell strainer. 5 Single cells were stained using FITC Annexin V Poptosis Detection Kit I (BD, USA) and analyzed on a flow cytometer (Beckman, Germany).

[0078] Electron Microscope

[0079] Cerebellar tissues of 6.5-month-old PDE9A-treated rats, littermate CHIP p.T246M homozygous mutant rats, littermate wild male rats, and 7.3-month-old CHIP overexpressing rats were collected and placed in electron microscopy tissue fixative (Solebo, China), post-fixed with 1% osmium acid (Ted Pella Inc, China) for 2 h, washed and dehydrated with gradient alcohol, infiltrated and embedded, polymerized, sectioned, and stained with 2% uranyl acetate saturated alcohol solution in the dark for 8 min; washed 3 times with 70% alcohol and ultrapure water, respectively, stained with 2.6% lead citrate solution in the dark for 8 min, washed 3 times with ultrapure water, and observed under a transmission electron microscope after drying.

[0080] Nissl stain

[0081] After dewaxing and antigen retrieval, tissue paraffin sections were stained using Nissl Stain Kit (Cresyl Violet Method) (Solebo, China).

[0082] Behavioral testing

[0083] Water maze experiment

[0084] The water maze hidden jump experiment was performed using SANS animal behavior analysis system software (Sansbio, China). A cylindrical barrel with a diameter of 150 cm and a height of 60 cm was selected, divided into the first quadrant (NE), the second quadrant (SE), the third quadrant (SW), and the fourth quadrant (NW). The liquid reached 40 cm and the temperature was maintained at 22 °C. A small cylinder with a diameter of 12 cm was placed vertically at a position close to the center circle of NW and deflected 30 degrees clockwise as an experimental platform. At the beginning of the experiment, the height of the experimental platform was adjusted to 1.5 cm above the liquid level, the exploration time of the water maze positioning and navigation experiment was set to 60 s, and the standing time on the platform was set to 5 s. Each rat entered the water maze from the NE quadrant to the barrel wall, and from the outer circle of the SE, SW, and NW quadrants in turn. From the first day to the fifth day, the experimental platform was immersed 1.5 cm below the horizontal plane. The rest of the operations were the same as before and re-recorded. On the 6th day, the experimental platform was removed, the exploration time was still set to 60 seconds, and the mice were still facing the barrel wall and placed in the water maze from NE, SE, SW to NW quadrants. The number of times the rat explored the platform and the ratio of the exploration time of the platform quadrant (NE) to the total time were recorded within 60 seconds to measure the rat's cognitive and memory abilities.

[0085] Wheel experiment

[0086] The motor coordination ability of rats was tested using a rotating wheel tester (Rot-Rod, Softmaze). The rats were trained for 4 consecutive days, three times a day, with an interval of 30 minutes each time, and each training reached a maximum speed of 40rpm / min. The test was conducted and recorded on the fifth day. The rats were placed on the rotating rod at an initial speed of 4rpm / min, and gradually accelerated to 40rpm / min within 5 minutes. The speed of the rotating rod corresponding to the second fall of the rat was recorded. If the axis-holding phenomenon occurred, it was counted as a fall if it occurred twice in total.

[0087] Balance beam experiment

[0088] Referring to previous experimental reports, a wooden stick with a length of 150 cm and a diameter of 18 mm was made, and the test height was 40 cm. Rats were trained to walk from the starting point to the end point without stopping three times a day, with an interval of 2 minutes each time. The training lasted for 4 consecutive days, and the time taken by the rats to walk the entire balance beam was recorded on the fifth day.

[0089] Gait analysis

[0090] A rat runway with a length of 2m, a width of 20cm, and a height of 20cm on both sides was placed on a horizontal experimental table. Clean white paper was spread on the bottom of the runway. Blue non-toxic dye was applied to the front feet of the rats, and red dye was applied to the hind feet. The rats were placed from the end of the runway. The gait trajectory of the rats from the end to the front was observed for two consecutive days. The gait trajectory was collected on the third day and calculated and analyzed.

[0091] in conclusion

[0092] 1. CHIP and PDE9A regulate each other's expression and interact with each other

[0093] PDE9A protein is composed of 593 amino acids, including a regulatory site at the amino terminal and a catalytic group at the carbon terminal. CHIP protein mainly plays a role in the protein quality control system through the TP R-Ubox dual domain. In order to explore the interaction between PDE9A protein and CHIP protein, we first exogenously increased the expression of PDE9A protein in cells. As the expression of PDE9A increased, the expression of CHIP protein gradually decreased ( Figure 1 a); On the contrary, as the expression of exogenous CHIP increased, the expression of PDE9A protein gradually decreased ( Figure 1 b); The results showed that there was a negative regulatory relationship between PDE9A and CHIP. After the CHIP mRNA expression changed, PDE9A mRNA did not change significantly ( Figure 1 c), and when the corresponding CHIP protein expression level changes, the PDE9A protein level immediately shows the opposite change ( Figure 1 d); Similarly, changes in PDE9A mRNA levels did not affect CHIP mRNA expression ( Figure 1 e), and when the corresponding PDE9A protein expression level changed, the CHIP protein expression level also showed the opposite change ( Figure 1 f); therefore, the mutual negative regulation between PDE9A and CHIP occurs post-transcriptionally. The half-life of CHIP protein is 6h. After the addition of protease inhibitor Bortzeomib, its half-life increased significantly, indicating that CHIP protein was hydrolyzed through the protease pathway; while the half-life of PDE9A protein was 4h. After the addition of autophagy inhibitor BafilomycinA1, its protein degradation was significantly delayed, suggesting that PDE9A protein was degraded through the autophagy pathway ( Figure 1 g). CHIP protein can promote the autophagic degradation of PDE9A, shortening the half-life of PDE9A to 2h. At the same time, PDE9A can enhance the proteolysis of CHIP protein, thus shortening the half-life of CHIP protein to 4h ( Figure 1 h, Figure 1 i), while the PDE9A inhibitor BAY 73-6691 prolongs the half-life of CHIP protein to 8h by promoting PDE9A degradation ( Figure 1 i). To further determine the binding domain of PDE9A protein and CHIP protein, a plasmid lacking the TPR domain and Ubox domain of CHIP protein was constructed ( Figure 1 g), and co-immunoprecipitation was performed after double transfection of 293T cells with PDE9A. The results showed that PDE9A could interact with the TPR domain of CHIP protein and bind to the U-box domain of CHIP protein, indicating that PDE9A protein may participate in the molecular chaperone activity of CHIP protein, and CHIP protein mainly interacts with PDE9A through the TPR structure ( Figure 1 k). In addition, immunofluorescence showed that CHIP protein and PDE9A protein were co-localized in the cytoplasm ( Figure 1 l).

[0094] CHIP protein is mainly composed of three major domains, TPR, GC and Ubox domains. After removing the TPR domain, neither GC nor Ubox domains interacted with PDE9A, while after removing the U-box domain, PDE9A interacted significantly with the TPR domain, indicating that PDE9A protein mainly binds to the TPR domain of CHIP protein. TPR is a simple repeating sequence composed of 34 amino acids, usually forming a helix-turn-helix folding structure. Each repeating sequence is linked by a short ring structure and grooves are formed inside it. These grooves become the main structure of TPR binding peptides. According to the structural characteristics of TPR protein, there are mainly five ways of binding between TPR structure and interacting proteins to mediate the interaction between proteins, including: short peptide binding to TPR groove, long peptide binding to TPR groove, TPR hinge formation and binding of folding domain to TPR protein. According to the Protein Data Bank (PDB), CHIP protein binds to the TPR groove through a short peptide, and the short peptide that forms this binding mechanism is controlled by a specific sequence, that is, the peptide is highly negatively charged (MEEVD) and must be located at the C-terminus. The most known TPR-binding short peptide is the co-chaperone protein Hsp70-Hsp90 organising protein (HOP). CHIP binds to HSP70 to mediate proteasome and lysosomal degradation of substrate proteins; while substrate proteins bind to CHIP-HSP90 and are only degraded through the proteasome. Since CHIP mediates the degradation of PDE9A by the autophagy-lysosomal pathway, PDE9A connects to the TPR domain of CHIP by binding to the chaperone protein HSP70. Hsp70 is one of the most common molecular chaperones that can prevent aggregation, promote the formation of native conformations, and dissolve and refold aggregated proteins. Hsp70 consists of a 45kDa N-terminal nucleic acid binding domain (NBD) and a 25kDa C-terminal substrate protein binding domain (SBD). The SBD contains a β-sandwich domain (β-SBD) of the substrate binding site and a flexible α-helical lid domain (α-SBD) that can regulate the affinity of misfolded proteins, indicating that PDE9A may directly bind to the β-SBD domain of HSP70 (Table 1):

[0095] Table 1. Prediction results of the position and distance of hydrogen bonds between PDE9A and HSP70

[0096]

[0097]

[0098] We used GRAMM-X software to perform docking between PDE9A (3QI4) and HSP70 (4B9Q), predicted 10 possible models, and visualized the top 1 predicted model in PDBePISA. The results showed that hydrogen bonds H accounted for the largest proportion in interfacing residues, indicating that PDE9A may form an H-bond link with HSP70 in the β-SBD domain ( Figure 2 ).

[0099] 2. CHIP mediates K63 and K27-linked ubiquitination of PDE9A

[0100] The Ubox domain at the C-terminus of the CHIP protein endows CHIP as an E3 ubiquitin ligase with important functions in substrate recognition, catalytic ubiquitin transfer, and determining the diversity and selectivity of ubiquitinated substrates. It can catalyze the connection of various substrate proteins with the seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) of ubiquitin molecules, leading to the formation of different polymerized ubiquitin chains, and then ubiquitination modification of substrate proteins after translation, thereby promoting ubiquitin-regulated substrate protein activity to regulate cell autophagy, mitochondrial function, lysosomal biogenesis and other corresponding biological processes. CHIP-mediated K48, K29 and K11 chain ubiquitination reactions activate protease degradation signals, K63 chain ubiquitination participates in the autophagic lysosomal degradation of proteins and the activity regulation of different signal proteins, while K27 chain ubiquitination mediates changes in mitochondrial function ( Figure 3 a).

[0101] Ubiquitination experiments were performed to explore how CHIP regulates PDE9A ubiquitination. In vitro ubiquitination experiments showed that PDE9A can be modified by CHIPE3 ligase as a ubiquitination substrate ( Figure 3 c), and the E2 conjugating enzymes Ube2V-Ubc13, UbcH5D and UbcH5a can recognize PDE9A together with CHIP protein and ubiquitinate it ( Figure 3 d). UbcH5a and UbcH5D have little selectivity in catalyzing the ubiquitination of PDE9A, while Ube2V1 / Ube2V2 locates ubiquitin K63 and catalyzes the formation of specific K63-linked ubiquitin chains. In addition, in vivo ubiquitination experiments of PDE9A and CHIP protein were performed in 293T cells, and the results also confirmed that PDE9A can be ubiquitinated by CHIP in vivo ( Figure 3 e), and then a series of Ub mutant plasmids were used to identify the ubiquitin lysine residues required for PDE9A to catalyze the formation of polyubiquitin chains. In the Ub plasmids with a single site retained, K48A, K63, K11, K27, and K29 ​​chains were all generated, while K11 ubiquitin chains were less synthesized ( Figure 3f, Figure 3 g), indicating that the above sites can all participate in the ubiquitination reaction of PDE9A regulated by CHIP. However, in the in vivo ubiquitination reaction in the Ub plasmid with single site inactivation, the ubiquitination reaction of K63R chain and K27R chain was obviously destroyed compared with the single site activity retention plasmid group, which again suggests that CHIP directly catalyzes the ubiquitination of K63 and K27 linked PDE9A. The full length of PDE9A protein is 593 amino acids in total, with a total of 30 arginine sites. Through the targeted E3 ligase prediction software (GPS-Uber), CHIP was specifically selected as the E3 ubiquitin ligase to analyze its possible ubiquitination site for PDE9A. The results predict that CHIP catalyzes the polyubiquitination of the 194th arginine site of PDE9A (Table 2).

[0102] Table 2. Prediction of CHIP ubiquitination sites on PDE9A

[0103]

[0104] Position: amino acid site, k: arginine; RING: RING-type E3 ligase

[0105] As an E3 ligase, CHIP has the ability to connect target proteins and specific E2s. Due to the presence of its specific TPR domain, its linkage with target proteins is diverse. Currently, there are three main ways in which CHIP links to target proteins: chaperone-mediated or misfolded protein degradation in cells: CHIP directly binds to substrate proteins to undergo ubiquitin-proteasome system (UPS)-mediated substrate protein degradation; the molecular chaperone HSP90-linked protease pathway and the molecular chaperone HSP70-dependent lysosomal pathway; how CHIP transfers ubiquitin to PDE9A and catalyzes the degradation of PDE9A. It has been previously confirmed that PDE9A interacts with the TPR domain, and CHIP promotes PDE9A to complete autophagy-lysosomal degradation, indicating that PDE9A protein links to CHIP by binding to HSP70. In addition, because both the CHIP TPR domain and the U-box domain interact with PDE9A, we need to further determine the specific details of CHIP ubiquitination modification of PDE9A. When we removed the CHIP protein TPR domain and U-box domain respectively, the CHIP protein ubiquitination reaction on PDE9A was affected. The CHIP TPR domain and U-box domain were changed, the molecular chaperone and E3 ligase functions were inactivated, and CHIP could not ubiquitinate PDE9A ( Figure 3h). Similarly, plasmids with CHIP protein T246M mutation and K30A mutation were transfected for in vivo ubiquitination, and the results were completely consistent with those after TPR domain deletion and U-box domain deletion. CHIP protein inactivation mutations can destroy CHIP ubiquitination PDE9A reaction ( Figure 3 b, Figure 3 i). The above results suggest that CHIP recruits K63 and K27 chains to transfer to PDE9A protein bound to HSP70, promoting tandem ubiquitination of PDE9A, and then completing the degradation of PDE9A through autophagy-lysosome.

[0106] 3. PDE9A inhibitor BAY 73-6691 and exogenous expression of CHIP protein can improve the symptoms of CHIP mutation animal models

[0107] Previous work in this study showed that the CHIP mutation rat model is consistent with human CHIP mutation cases, showing progressive ataxia and cerebellar neuron degeneration and death. At the same time, the expression of PDE9A protein in the animal model brain increased significantly, and its expression increased progressively with the progression of the disease. This result suggests that upregulation of PDE9A protein expression may be one of the important causes of CHIP mutation, but the mechanism is still unclear. Bay73-6691 is a PDE9A inhibitor. In the past, it has significantly improved memory loss by increasing retention of long-term potentiation (LTP), enhancing hippocampal synaptic plasticity, and reducing the damage of toxic protein aggregation to hippocampal neurons. It is mainly used in the study of improving symptoms such as memory loss and cognitive impairment in Alzheimer's disease.

[0108] Based on previous work, we speculated whether PDE9A inhibition might have a therapeutic effect on CHIP mutant animal models? In further experiments, we selected 5.5-month-old T246M homozygous mutant male mice and injected them intraperitoneally with Bay73-6691 200ug / kg for 15 consecutive days. After 6 months of age, we compared them with untreated homozygous mutant male mice from the same litter, wild male mice from the same litter, and 4-month-old homozygous mutant male mice without clinical symptoms for behavioral testing ( Figure 4 a), the test results confirmed that after Bay73-6691 treatment, the balance and coordination ability and gait of rats were alleviated, including the tolerance of Bay group rats in the wheel test ( Figure 4 b); The time required to cross the balance beam is shortened ( Figure 4 c); and the gait spacing increases and the uniformity improves ( Figure 4 de), In addition, the rats' memory was significantly improved, and the time to find the platform in the water maze test was shortened ( Figure 4fg), the percentage and number of platform crossings increased significantly ( Figure 4 hi). These results suggest that Bay73-6691 can treat the clinical phenotype of autosomal recessive spinocerebellar ataxia by inhibiting the toxic damage mediated by PDE9A protein aggregation.

[0109] The inactivation mutation at the p.T246M site of the STUB1 gene leads to the inactivation of CHIP protein. The expression of CHIP protein in brain tissue is reduced. The reduction of CHIP protein can present the typical clinical manifestations of cerebellar ataxia caused by SCAR: Therefore, we also gave exogenous CHIP gene expression as a positive treatment control. In the experiment, we also injected 1.2*10 AAV virus carrying CHIP gene that can pass through the blood-brain barrier into the caudal vein of 5.5-month-old rats. 11 vg / rat. After CHIP protein was completely overexpressed in the brain tissue of 7-month-old rats, behavioral observations showed that the balance and coordination ability and gait of the CHIP overexpression group rats were alleviated compared with the homozygous mutant rats in the same litter ( Figure 4 a, Figure 4 jm), memory and cognitive abilities significantly improved ( Figure 4 nq). This indicates that CHIP protein has a significant improvement effect on ataxia gait and cognitive memory impairment. The above results suggest that both BAY73-6691 treatment and exogenous CHIP expression gene therapy have therapeutic effects on CHIP mutation models.

[0110] 4. PDE9A inhibits PKG to directly phosphorylate CHIP, and the PDE9A inhibitor BAY 73-6691 increases the phosphorylation level and expression of CHIP protein

[0111] Recent studies have found that protein kinase G (PKG), as the main effector of the second messenger cGMP, can directly activate the phosphorylation of the CHIP-S19 (human, mouse S19) site located in the highly conserved residues in the TPR domain, which increases the binding affinity of CHIP to Hsp70 and reduces the self-degradation of CHIP by proteases after ubiquitination, thereby prolonging the half-life of CHIP protein. Subsequently, PKG mediates CHIP-pS19 protein to better clear ubiquitin-insoluble proteins. We speculate whether PDE9A reduces CHIP protein expression and reduces the half-life of CHIP protein in vivo by regulating the changes in CHIP phosphorylation levels; whether PDE9A inhibitors can inhibit PDE9A levels to increase CHIP protein phosphorylation and thereby enhance the binding of CHIP to HSP70, further promoting the binding of PDE9A protein to CHIP-HSP70 and ubiquitination degradation.

[0112] The CHIP-S20 site-specific phosphorylation antibody was used to detect the cerebellum and hippocampus of rats with CHIP p.T246M inactivation mutation, which showed a decrease in p-CHIP protein. After inhibiting PDE9A with Bay-736691, the phosphorylation level of CHIP protein S20 was significantly increased ( Figure 5 a), while exogenously increasing CHIP protein levels, p-CHIP increased ( Figure 5 b), in vitro cell experiments also confirmed that p-CHIP expression decreased after CHIP Ubox inactivation mutation, and P-CHIP levels decreased significantly with increased PDE9A protein or decreased CHIP protein ( Figure 5 c). Since PDE9A does not have a cGMP binding region, the catalytic activity of PDE9A is not enhanced by cGMP, so PDE9A inhibitors increase PKG levels by increasing baseline cGMP concentrations. Therefore, PDE9A reduces phosphorylated CHIP protein by reducing PKG content, reducing the half-life of CHIP protein and increasing the hydrolysis of CHIP protein.

[0113] 5. PDE9A inhibitor BAY 73-6691 treatment improves CHIP mutation mitochondrial autophagy disorder and cell apoptosis

[0114] PDE9A regulates CHIP phosphorylation changes through cGMP-PKG signaling to reduce the expression of CHIP protein in vivo. The reduction of CHIP protein inhibits the k63 chain and K27 chain tandem ubiquitination levels of PDE9A, inhibits the lysosomal degradation of PDE9A, and increases the aggregation of intracellular PDE9A. Previous results showed that rats with CHIP p.T246M site mutation mainly had pathological damage to cerebellar Purkinje cells, nuclear condensation, dendritic swelling, and a significant reduction in the number of Purkinje cells compared with normal wild rats; we inferred that increased aggregation of PDE9A and reduced CHIP protein can both lead to cell destruction and apoptosis. Therefore, the AnnexinV / PI flow apoptosis experiment was used to detect the apoptosis of PDE9A and CHIP protein abnormalities. The results showed that the CHIP p.T246M mutation, reduced CHIP protein, and increased PDE9A protein could all lead to varying degrees of apoptosis in 293T cells, while after treatment with BAY 73-66791 or increasing CHIP protein expression, the level of cell apoptosis was significantly reduced ( Figure 6 a), Similarly, in the CCK8 cell experiment, it was found that the increase of PDE9A and CHIPp.T246M mutation significantly reduced the cell proliferation activity, while after BAY 73-66791 treatment or the increase of CHIP protein, the proliferation activity of 293T cells was significantly improved ( Figure 6b), therefore, the increased aggregation of PDE9A and the reduction of CHIP protein caused by CHIP p.T246M mutation can reduce cell activity and affect normal cell proliferation. On the other hand, tissue immunofluorescence observation showed that after BAY 73-66791 treatment, the Purkinje marker protein calbindin in the cerebellum of CHIP p.T246M mutant rats increased significantly, and the number of Purkinje neurons increased ( Figure 7 a); After exogenous addition of CHIP protein, the number of Purkinje neurons increased significantly and the number of neuronal dendrites was significantly improved compared with the CHIP p.T246M mutant rats of the same littermate and age ( Figure 7 b); Nissl staining of the hippocampus of rats with CHIPp.T246M mutation showed that the number of neurons in mutant rats was reduced and their arrangement was disordered. After treatment with BAY 73-66791 and the addition of exogenous CHIP protein, the number and arrangement of neurons improved ( Figure 8 ).

[0115] CHIP plays an important role in mitochondria. Mitochondria lacking CHIP can show changes in oxidative stress. When CHIP has a complete TPR domain and E3 ubiquitin ligase activity, CHIP regulates the autophagy level of damaged mitochondria (mitophagy); in addition, the decrease in the number of CHIP inactivation increases the number of abnormal mitochondria associated with CHIP by inhibiting the LC3B-mediated autophagy reaction30. CHIP-mediated K63-linked polyubiquitin chains often modify substrates for degradation in the autophagy-lysosome pathway, and play a complementary protective role in maintaining protein homeostasis by directing abnormal proteins and organelles to autophagosomes. K27-linked ubiquitin chains mainly mediate the autophagy process, which can be recognized by the autophagy receptor P62 and form autophagosomes, ultimately inducing mitophagy and alleviating oxidative stress. The formation of tandem ubiquitin chains connected by K63 and K27 mainly promotes autophagy to selectively degrade aggregated proteins and non-functional mitochondria, and initiates the signal cascade to activate mitophagy; PDE9 can be localized in mitochondria, and inhibiting its activity can activate the oxidative respiration reaction of mitochondria and reduce mitochondrial swelling. Therefore, we want to know whether the loss of CHIP E3 ligase activity mediates the formation of K63 and K27 chain ubiquitination of PDE9A, and whether the reduction and loss of CHIP function accompanied by increased PDE9A aggregation will lead to abnormal mitochondrial functional structure and disruption of mitochondrial autophagy balance. p62, also known as SQSTM1, can connect LC3 and ubiquitinated substrates, which are then incorporated into autophagosomes and degraded in autophagic lysosomes. When autophagy is inhibited, autophagosomes accumulate, p62 levels increase, and LC3B decreases. Coincidentally, in rats with CHIP U-box inactivation mutations, P62 increased and LC3B decreased in the hippocampus and cerebellum tissues, while in male rats of the same littermate, after treatment with BAY 73-6691 and increased CHIP protein, P62 decreased while LC3B expression increased ( Figure 7 c, Figure 7 d); Further transmission electron microscopy revealed that compared with rats with CHIP U-box inactivation mutation, the number of mitochondria in the cerebellum tissue was relatively small, the membrane was partially blurred and damaged, the cristae structure was largely broken and dissolved, the matrix was largely dissolved or vacuolated, and autophagosome structures were visible under the microscope; the number of mitochondria in the cerebellum tissue of rats after adding PDE9A inhibitors and increasing the CHIP protein content in brain tissue was abundant, the size was uneven, and the membrane was relatively intact ( Figure 7 e). This suggests that the reduction and loss of CHIP function accompanied by increased PDE9A aggregation damages mitochondrial structure and function, inducing mitophagy disorders; while BAY 73-66791 inhibits PDE9A aggregation and increases CHIP protein expression to improve mitochondrial damage and maintain mitophagy balance.

[0116] 6. Single-cell sequencing results suggest that dysregulation of cGMP and cAMP signaling pathways is the main pathway change in CHIP mutations

[0117] Studies have shown that CHIP mutation SCAR mainly manifests as spinocerebellar ataxia and cognitive impairment, suggesting that the cerebellum and the hippocampus, which are closely related to cognition, are closely related to the disease. Single-cell sequencing technology (ScRNA) can reveal the pathological changes of the disease at the single cell level. Therefore, in order to clarify the regulatory relationship between PDE9A-CHIP at the single cell level, this study performed single-cell sequencing on the hippocampus and cerebellum tissues of the rat disease model.

[0118] We obtained four groups of scRNA results, including CHIP mutant group (Hom), CHIP overexpression group (AAV-CHIP), PDE9A treatment group (Bay) and normal male rat group (Wt), capturing 19279, 25238, 23177 and 13473 single cells, respectively. Fig. 9 a). The expression of the root marker genes divided 81,167 single cells into 12 cell subpopulations, including granule cells, oligodendrocytes, glutamatergic neurons, GABAergic neurons, GABAergic interneurons, pyramidal cells, oligodendrocyte precursor cells, endothelial cells, Bergmann glia, microglia, Purkinje cells, and astrocytes ( Fig. 9 bc). Heatmaps show that each cell subset expresses specific genes.

[0119] KEGG enrichment analysis of differentially expressed genes in all cell subpopulations found that the differentially expressed genes in the Hom group compared with the Wt group were enriched in the cAMP signaling pathway, cGMP-PKG signaling pathway ( Fig.10 a). AUCell and GSEA analysis found that compared with the Wt group, the Hom group showed significant inhibition of the cGMP-PKG signaling pathway and significant activation of the cAMP signaling pathway ( Fig.10 be).

[0120] Purkinje cells are the main neuronal subpopulation affected by CHIP mutations. We further explored the changes in Purkinje neurons using single-cell sequencing. Through cell ratio analysis, we found that the proportion of Purkinje cells in the Hom group was significantly lower than that in the Wt group, while the proportion of Purkinje cells increased after Bay and AAV-CHIP treatment, suggesting that CHIP mutations can lead to a decrease in the number of Purkinje cells, and that Bay and AAV-CHIP treatment can restore the number of Purkinje cells and improve symptoms to a certain extent. Similar to the overall cell analysis, the results of KEGG enrichment analysis of differentially expressed genes in Purkinje cells showed that the differentially expressed genes in the Hom group compared with the WT group were also enriched in ubiquitin-mediated proteolysis, cAMP signaling pathway, spinocerebellar ataxia, and cGMP-PKG signaling pathway ( Fig.11 a).

[0121] Previous studies have shown that CHIP has a regulatory effect on the cAMP-PKA pathway. The PKA holoenzyme is composed of two catalytic subunits (PKAc) and two regulatory subunits (PKAr). The PKA holoenzyme is inactive. Only after activation by the second messenger cAMP, the conformation of the regulatory subunits is changed to release the active subunit PKAc27. The catalytic subunit α of protein kinase A (PRKACA) is the main subtype of ppkac, which is expressed in most tissues and is mainly used to present the activity status of PKAc in tissues.

[0122] Previous studies have reported that CHIP can mediate the ubiquitination and degradation of PKAc. To verify the results of single-cell sequencing, we detected increased expression of PRKACA in the cerebellum and hippocampus of CHIP mutant rats ( Fig.12 ab), we further added Forskolin to the ubiquitination reaction of PDE9A and CHIP in vivo to increase cAMP levels by activating adenylate cyclase. The results showed that with the increase of cAMP concentration, CHIP-mediated K63-chain and K27-chain linked PDE9A ubiquitination decreased, suggesting that cAMP-induced PKAc upregulation may compete with PDE9A for CHIP ubiquitination and inhibit PDE9A degradation ( Fig.12 c).

[0123] 7. AAV-CHIP and PDE9A inhibitor BAY 73-6691 participate in the bidirectional regulation of cGMP and cAMP signals and play a therapeutic role

[0124] In order to clarify whether overexpression of CHIP can rescue the pathological changes caused by its mutation, we found through monocyte sequencing that overexpression of CHIP can eliminate the pathway abnormalities caused by CHIP mutation, that is, compared with the AAV-CHIP group, the activity of the cGMP-PKG signaling pathway in the Hom group was significantly downregulated, while the activity of the cAMP signaling pathway was significantly activated ( Fig.10 bc, Fig.10 fh). At the same time, the Purkinje cell pathway activity analysis also supported the above results ( Fig.11 b, Fig.11 de). In conclusion, the results once again confirmed that CHIP can activate the cGMP / PKG signaling pathway and inhibit the cAMP signaling, thereby maintaining the intracellular cGMP / cAMP homeostasis.

[0125] It is known that CHIP mutations can lead to abnormal formation of K63 and K27 polyubiquitin chains on PDE9A, thereby affecting the degradation of PDE9A in autophagic lysosomes. Moreover, PKG can mediate the reduction of p-CHIP and the reduction of CHIP half-life caused by the increase of PDE9A. In view of the above-mentioned role of PDE9A in CHIP mutation and the fact that the BAY 73-6691 inhibitor can reduce PDE9A activity, we speculate that BAY 73-6691 may have ARCA therapeutic effects after CHIP mutation. Therefore, we performed monocyte sequencing on the Hom group treated with BAY 73-6691. The results were consistent with expectations. The Bay group showed a significant therapeutic effect on the Hom group, just like overexpressing CHIP ( Fig.10 c, Fig.10 ik). The results of Purkinje cell analysis showed that the differentially expressed genes in the Bay group compared with the Hom group were mainly enriched in oxidative phosphorylation, cAMP signaling pathway and induced proteolytic reactions ( Fig.11 c), supporting the involvement of PDE9A in CHIP-mediated ubiquitination, and CHIP may be involved in PDE9A-mediated phosphorylation modification. It is worth noting that the results of Aucell analysis showed that the decrease in CHIP's E3 ubiquitin ligase activity led to a decrease in CHIP expression levels, which in turn affected the proteolytic reaction induced by CHIP as an E3 ligase, while increasing intracellular cAMP activity. The activation of the ubiquitin-mediated proteolytic reaction in which CHIP participated was inhibited, and the decrease in PDE9A promoted autophagy and mitochondrial autophagy ( Fig.11 fh). These results support that CHIP promotes the degradation of PDE9A in autophagolysosomes through K63 and K27 chain ubiquitination modification, while cAMP can competitively inhibit the ubiquitination of PDE9A. In addition, after BAY 73-6691 treatment, the cGMP-PKG signaling pathway and oxidative phosphorylation were significantly activated ( Fig.11 c, Fig.11 i), supporting that PDE9A inhibits CHIP-S19 phosphorylation and reduces the levels of CHIP and p-CHIP in Purkinje cells.

[0126] In order to comprehensively evaluate the effects of CHIP mutation, CHIP overexpression and BAY 73-6691 treatment on animal models, we found that the genes in the cAMP signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation, ubiquitin-mediated proteolysis, mitochondrial autophagy and autophagy pathways changed significantly in the heat map. In addition, compared with the Hom group, the wild type, CHIP overexpression group and Bay treatment group showed high consistency. These results are highly consistent with previous conclusions, once again confirming that PDE9A and CHIP are potential therapeutic targets for ARCA.

[0127] It is known that cGMP, PKG1 and PKG2 are key molecules in the cGMP-PKG signaling pathway, while cAMP and PRKACA are key molecules in the cAMP signaling pathway. In order to verify the accuracy of the above conclusions, we further detected these key molecules in the cerebellum and hippocampus of each group of rats using the WB method. The results showed that CHIP, cGMP, PKG1 and PKG2 decreased in the Hom group, while PDE9A increased. After treatment with overexpression of CHIP and Bay, the levels of the above proteins were reversed ( Fig.12 dg); In addition, cAMP and PRKACA increased significantly in the Hom group, but this increase was downregulated with the increase of CHIP protein and the inhibition of PDE9A ( Fig.12 ab), which once again confirmed that CHIP ubiquitination reduced the expression of PKAc in CHIP-inactive mutant animals, and PKAc competitively increased PDE9A protein. In summary, the above results effectively suggested that CHIP could mediate the interaction between cGMP and cAMP signals by reducing the affinity of PDE9A for hydrolyzing cGMP.

[0128] At the same time, we compared the differences in pathogenic pathways between other cell subpopulations and Purkinje cells. The results showed that the differential genes of five types of cell subpopulations, including granule cells, Bergmann glial cells, glutamatergic neurons, oligodendrocyte precursor cells, and oligodendrocytes, were significantly enriched in the cAMP signaling pathway and the cGMP-PKG signaling pathway, showing pathway disorders similar to those of Purkinje cells. Although overexpression of Bay and CHIP can eliminate abnormal changes in some cell subpopulations, AUCell analysis only supports similar changes in granule cells and Purkinje cells in the above pathways, while these changes are not significant in other subpopulations, suggesting that different biological mechanisms may be involved.

[0129] In summary, the results of monocyte sequencing not only support that CHIP mutation leads to abnormal formation of K63 and K27 polyubiquitin chains on PDE9A and affects the degradation of PDE9A in autophagic lysosomes, but also that PKG mediates the increase of PDE9A leading to a decrease in p-CHIP and a decrease in CHIP half-life. At the same time, it also confirms that CHIP overexpression and BAY 73-6691 can improve the imbalance of the intracellular cGMP / cAMP pathway, thereby having a potential therapeutic and brain-protective effect on ARCA.

[0130] Implementation effect example

[0131] The ability of CHIP to enhance the clearance of damaged / misfolded proteins and inhibit proteotoxicity is associated with immunity, aging, metabolic stress, and many human diseases, including cardiac and neurological diseases. Bi-allelic CHIP mutations are the direct cause of ARCA. Here, we revealed that upregulation of PDE9A protein expression is an important pathological mechanism of CHIP mutations. CHIP and PDE9A mutually regulate expression and interact with each other, and their interaction is mediated by the TPR domain of CHIP. The CHIP TPR domain may bind to Hsp70 to link PDE9A, and the CHIP U-box domain recruits K63 chains and K27 chains to ubiquitinate PDE9A, mediating the autophagic lysosomal degradation of PDE9A.

[0132] After CHIP mutation, PDE9A cannot be degraded by autophagy and instead aggregates normally in the cell; PDE9A protein increases, hydrolyzes cGMP, inhibits PKGs S20 site phosphorylation modification of CHIP and reduces the half-life of CHIP protein, resulting in a decrease in CHIP protein. At the same time, CHIP ubiquitination modification of PKAc is weakened, feedback enhances the cAMP signaling pathway, and upregulation of the cAMP pathway further increases the level of PKAc to competitively inhibit PDE9A degradation through ubiquitination; abnormal aggregation of PDE9A increases, and reduction of CHIP protein can disrupt the balance of mitochondrial autophagy in cells, ultimately leading to cell death. When PDE9A activity is inhibited by the PDE9A inhibitor BAY 73-6691, the above pathological outcomes can be improved. Given the important role of CHIP in cardiac, skeletal muscle, neurogenesis, and tumor diseases, our results have therapeutic significance, revealing that PDE9A inhibitors are a potential promising therapeutic drug for CHIP mutation-related ataxia.

[0133] Our study also found that CHIP plays an important role in maintaining the balance of cAMP and cGMP. Using single-cell sequencing technology, we found that in the CHIP mutation model, the cGMP-PKG signal interaction was inhibited, while the cAMP signaling pathway was activated. Exogenous overexpression of CHIP or the use of PDE9A inhibitors can upregulate CHIP expression, increase cGMP, PKG1 and PKG2, and decrease PDE9A. With the increase of CHIP protein and the inhibition of PDE9A activity, cAMP and PKA were significantly downregulated. Our results suggest that the interaction between CHIP and PDE9A is a potential connecting molecule for bidirectional regulation of the cGMP and cAMP pathways ( Fig.13 ).

[0134] The signal interaction of intracellular second messengers cAMP and cGMP plays a very important role in cell signal transduction to maintain normal biological functions. cAMP can stimulate protein kinase A (PKA), open calcium ion channels, start cAMP-activated enzyme exchange protein (Epac), and activate downstream pathways; cGMP, as a second messenger, induces PKG, and its expression is limited to vascular tissues, lungs and brain tissues. In neuronal cells, cAMP and cGMP can mediate antagonistic cell behaviors caused by some in vitro factors, such as ion channels, cell volume regulation, and axon guidance. Local cAMP and cGMP in the undifferentiated axons of nerve cells can promote and inhibit the formation of axons, respectively, and in the process of dendrite formation, the two also play opposite roles; these two signal pathways antagonize and restrict each other, and jointly regulate the normal physiological functions of cells. In cardiomyocytes, it was found that the hydrolysis activity of cAMP is regulated by cGMP. In PDE2, cGMP stimulates the hydrolysis of cAMP through all osteric stimulation; in PDE3, cGMP competitively inhibits the hydrolysis of cAMP; however, PDE9A is a specific selective inhibitor of cGMP, which only selectively hydrolyzes cGMP and inhibits the cGMP / PKG pathway. Therefore, CHIP is crucial for PDE9A to bidirectionally regulate cGMP and cAMP in neurons, mediating the interaction between the two. BAY 73-6691 inhibits PDE9A activity to maintain the balance of cGMP / cAMP signal interaction mediated by CHIP and improve the pathological changes of ARCA brain injury caused by mitochondrial autophagy disorders in Purkinje cells.

[0135] The pathological manifestations of ARCA are diverse, the most common of which are neuronal atrophy and degeneration, loss of myelin and disappearance of Purkinje cells, and glial proliferation, resulting in extensive degeneration of the cerebellar hemispheres, vermis, and middle and inferior cerebellar peduncle; therefore, we first confirmed in the ARCA animal model that CHIP and PDE9A in Purkinje cells can be therapeutic targets for ARCA. In order to further explore the pathological changes of ARCA mediated by CHIP, monocyte subgroup analysis was used to observe the molecular changes of different cell types in the cerebellum and hippocampus. Although we found that granule cells and Purkinje cells have similar pathogenic mechanisms and can be eliminated by BAY73-6691 and CHIP overexpression. Granule cells are widely distributed in the hippocampus and cerebellum, and play an important role in maintaining hippocampal cognitive function and cerebellar balance function. Therefore, we believe that neuronal abnormalities such as granule cells and Purkinje cells play a major role in ARCA caused by CHIP mutations, which provides new supplements to the results of previous studies. In addition, several glial cells such as Bergmann glia, oligodendrocyte precursor cells, oligodendrocytes, and glutamatergic neurons showed different pathway activity from that of Purkinje cells, suggesting that different pathological mechanisms may be involved. These findings may be related to the pathogenesis of other CHIP-mediated diseases previously reported.

[0136] In recent years, PDE9 inhibitors have been widely used in research related to neurodegenerative diseases. Among them, PF-04447943 has entered Phase II clinical trials for Alzheimer's disease; BI-409306 has entered Phase II clinical trials for Alzheimer's disease and Phase II clinical trials for schizophrenia, while the earlier discovered PDE9A inhibitor Bay 736691 is still in the early stages of preclinical research. It is not clear whether BAY 73-6691 is suitable for human trials or whether it is still just a laboratory research tool. This study confirmed that the PDE9A inhibitor BAY 73-6691 can improve neurological damage in CHIP mutation models through the dual pathway of ubiquitin phosphorylation, and may become a candidate drug for the treatment of CHIP-related diseases, providing a new method for the treatment of related diseases, especially CHIP mutation ataxia.

[0137] Therefore, it is obvious to those skilled in the art that pharmaceutically acceptable salts of BAY 73-6691 can also achieve the above technical effects. The salt in the pharmaceutically acceptable salt of BAY 73-6691 is any one of hydrochloride, hydrobromide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, propionate, hexanoate, heptanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, maleate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, p-toluenesulfonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, dodecyl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate and stearate.

[0138] The above acids are all common acid substances in the human body, so the salts containing these acids are easily absorbed by the human body and then exert their effects.

[0139] This study also suggests that the PDE9A inhibitor BAY 73-6691 regulates the cGMP / cAMP pathway in the central nervous system and is involved in regulating the CHIP-related protein quality control system, explaining the possible new drug mechanism and target of the PDE9A inhibitor BAY 73-6691.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a pharmaceutical preparation containing BAY 73-6691 or its derivatives in the preparation of a drug for treating brain injury diseases, wherein the structural formula of BAY 73-6691 is: .

2. The use according to claim 1, characterized in that: The pharmaceutical preparation comprises BAY 73-6691 and / or a pharmaceutically acceptable salt of BAY 73-6691.

3. The use according to claim 2, characterized in that: The salt in the pharmaceutically acceptable salt of Bay73-6691 is any one of hydrochloride, hydrobromide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, propionate, hexanoate, heptanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, maleate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, p-toluenesulfonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, dodecyl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate and stearate.

4. The use according to any one of claims 1 to 3, characterized in that: The brain injury disease is ARCA-induced brain injury.

5. The use according to any one of claims 1 to 3, characterized in that: The brain damage disease is autosomal recessive spinocerebellar ataxia.

6. The use according to claim 4, characterized in that: The ARCA-induced brain damage is caused by CHIP inactivating mutations.

7. The use according to claim 5, characterized in that: The CHIP inactivation mutation leads to increased PDE9A expression in ARCA brain tissue, and the PDE9A content increases progressively as the disease progresses.

8. The use according to claim 4, characterized in that: The autosomal recessive spinocerebellar ataxia is caused by a disorder of mitochondrial autophagy in Purkinje cells.

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